Interactions of external and internal H+ and Na+ with Na+/Na+ and Na+/H+ exchange of rabbit red cells: Evidence for a common pathway

Abstract

We have studied the kinetic properties of rabbit red cell (RRBC) Na+/Na+ and Na+/H+ exchanges (EXC) in order to define whether or not both transport functions are conducted by the same molecule. The strategy has been to determine the interactions of Na+ and H+ at the internal (i) and external (o) sites for both exchanges modes. RRBC containing varying Nai and Hi were prepared by nystatin and DIDS treatment of acid-loaded cells. Na+/Na+ EXC was measured as Nao-stimulated Na+ efflux and Na+/H+ EXC as Nao-stimulated H+ efflux and delta pHo-stimulated Na+ influx into acid-loaded cells. The activation of Na+/Na+ EXC by Nao at pHi 7.4 did not follow simple hyperbolic kinetics. Testing of different kinetic models to obtain the best fit for the experimental data indicated the presence of high (Km 2.2 mM) and low affinity (Km 108 mM) sites for a single- or two-carrier system. The activation of Na+/H+ EXC by Nao (pHi 6.6, Nai less than 1 mM) also showed high (Km 11 mM) and low (Km 248 mM) affinity sites. External H+ competitively inhibited Na+/Na+ EXC at the low affinity Nao site (KH 52 nM) while internally H+ were competitive inhibitors (pK 6.7) at low Nai and allosteric activators (pK 7.0) at high Nai. Na+/H+ EXC was also inhibited by acid pHo and allosterically activated by Hi (pK 6.4). We also established the presence of a Nai regulatory site which activates Na+/H+ and Na+/Na+ EXC modifying the affinity for Nao of both pathways. At low Nai, Na+/Na+ EXC was inhibited by acid pHi and Na+/H+ stimulated but at high Nai, Na+/Na+ EXC was stimulated and Na+/H+ inhibited being the sum of both pathways kept constant. Both exchange modes were activated by two classes of Nao sites, cis-inhibited by external Ho, allosterically modified by the binding of H+ to a Hi regulatory site and regulated by Nai. These findings are consistent with Na+/Na+ EXC being a mode of operation of the Na+/H+ exchanger. Na+/H+ EXC was partially inhibited (80-100%) by dimethyl-amiloride (DMA) but basal or pHi-stimulated Na+/Na+ EXC (pHi 6.5, Nai 80 mM) was completely insensitive indicating that Na+/Na+ EXC is an amiloride-insensitive component of Na+/H+ EXC. However, Na+ and H+ efflux into Na-free media were stimulated by cell acidification and also partially (10 to 40%) inhibited by DMA; this also indicates that the Na+/H+ EXC might operate in reverse or uncoupled modes in the absence of Na+/Na+ EXC.(ABSTRACT TRUNCATED AT 400 WORDS)